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The Use of Eosin B to Assess the Viability and Developmental Potential of Rat Embryos " (1988)

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The Use of Eosin B to Assess the Viability and Developmental Potential of Rat Embryos Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1988 The seu of eosin B to assess the viability and developmental potential of rat embryos Michael Patrick Dooley Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, Animal Sciences Commons, Physiology Commons, and the Veterinary Physiology Commons Recommended Citation Dooley, Michael Patrick, "The use of eosin B to assess the viability and developmental potential of rat embryos " (1988). Retrospective Theses and Dissertations. 8839. https://lib.dr.iastate.edu/rtd/8839 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS The most advanced technology has been used to photo­ graph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are re­ produced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. These are also available as one exposure on a standard 35mm slide or as a 17" x 23" black and white photographic print for an additional charge. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 8909140 The use of eosin B to assess the viability and developmental potential of rat embryos Dooley, Michael Patrick, Ph.D. Iowa State University, 1988 Copyright ©1988 by Dooley, Michael Patrick. All rights reserved. UMI 300N.ZeebRd. Ann Arbor, MI 48106 The use of eosln B to assess the viability and developmental potential of rat embryos by Michael Patrick Dooley A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department; Veterinary Physiology and Pharmacology Major: Physiology Approved: Members of the Committee: Signature was redacted for privacy. Signature was redacted for privacy. In Charge f Maioi WeTR Signature was redacted for privacy. 'For the Major Department Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1988 Copyright ® Michael Patrick Dooley, 1988. All rights reserved. il TABLE OF CONTENTS Page ABSTRACT x INTRODUCTION 1 Prelmplantatlon Period of Embryonic Development 1 Assessment of Frelmplantation Embryonic Viability 3 Background and Review of Literature 6 Morphological evaluation 6 In vitro development 10 In vivo development 14 Metabolic studies 16 Precursor uptake 16 Metabolic by-product 18 Isotope release 19 Dye assays 20 Dye metabolism 20 Vital staining 21 Nonvital staining 25 Dye-exclusion assays 27 Embryonic and/or maternal factors 28 STATEMENT OF THE RESEARCH PROBLEM 30 RESEARCH OBJECTIVE 31 MATERIALS AND METHODS 33 Animals 33 Strains utilized 33 Selection criteria 34 ill Page Care and maintenance 34 Mycoplasma detection and routine medication 35 Vaginal Smear, Mating, and Assessment of Mating 37 Vaginal smear 37 Mating and assessment of mating 38 Induction of pseudopregnancy 40 Media for Flushing and Recovery of Embryos 40 Embryo Retrieval and Handling 43 Embryo Evaluation and Assignment to Treatment 44 Embryo Staging 45 Conditions for Embryo Culture 47 Dye Formulation and Evaluation of Staining 48 Dye formulation 48 Evaluation of the staining response 48 Embrj'o Transfer, Monitoring of Pregnancy, and Determination of Embryo Survival 49 Embryo transfer 49 Monitoring of pregnancy 51 Determination of embryo survival 52 Statistical Analysis and Criteria for Significance 52 Experiment 1. Determination of the Minimal Temperature Needed to Block Development and Induce Staining of 4- to 16-Cell Rat Embryos when Exposed to Eosin B 53 Experimental objectives 54 Experimental design and procedure 54 iv Page Statistical analysis 56 Experiment 2. Influence of Stage of Embryonic Development on the Staining Response of Rat Embryos Exposed to 55"C 56 Experimental objective 57 Experimental design and procedure 57 Statistical analysis 57 Experiment 3. Effect of the Concentration of Eosln B and the Stage of Embryonic Development on the Time Required to Stain Dead Embryos 58 Experimental objective 58 Experimental design and procedure 58 Statistical analysis 60 Experiment 4. Effect of a Single Exposure to IX Eosln B on the In Vitro Development of Rat Embryos 60 Experimental objective 60 Experimental design and procedure 61 Statistical analysis 62 Experiment 5. Effect of a Single Exposure to 5X Eosln B on the In Vitro Development of Rat Embryos 63 Experimental objective 63 Experimental design and procedure 64 Statistical analysis 65 Experiment 6. Effect of Multiple Exposures to IX Eosln B on the In Vitro Development of Rat Embryos 66 Experimental objective 66 V Page Experimental design and procedure 66 Statistical analysis 68 Experiment 7, Effect of Continuous Exposure to IX or 5X Eosln B on the In Vitro Development of Rat Embryos 69 Experimental objective 69 Experimental design and procedure 70 Statistical analysis 71 Experiment 8. Viability of Control or of Rat Embryos Exposed Once to 5X Eosln B and then Transferred to the Left Horn of Naturally Mated Rats 72 Experimental objective 74 Experimental design and procedure 74 Statistical analysis 76 Experiment 9. Viability of Control or of Rat Embryos Exposed Once to 5X Eosln B and then Transferred to Both Horns of Naturally Mated Rats 76 Experimental objective 77 Experimental design and procedure 77 Statistical analysis 79 Experiment 10. Viability of Control or of Rat Embryos Exposed Once to 5X Eosln B and then Transferred to Opposite Horns of the Same Fseudopregnant Rat 80 Experimental objective 80 Experimental design and procedure 80 Statistical analysis 82 vi Page Experiment 11. Viability of Control or of Rat Embryos Exposed Once to 5X Eosin B and then Transferred to Both Horns of Fseudopregnant Rats 83 Experimental objective 84 Experimental design and procedure 84 Statistical analysis 85 RESULTS 87 Experiment 1. Determination of the Minimal Temperature Needed to Block Development and Induce Staining of 4- to 16-Cell Rat Embryos when Exposed to Eosin B 87 Experiment 2. Influence of Stage of Embryonic Development on the Staining Response of Rat Embryos Exposed to 55"C 93 Experiment 3. Effect of the Concentration of Eosin B and the Stage of Embryonic Development on the Time Required to Stain Dead Embryos 94 Initial staining 94 Complete staining 97 Experiment 4. Effect of a Single Exposure to IX Eosin B on the In Vitro Development of Rat Embryos 102 Experiment 5. Effect of a Single Exposure to 5X Eosin B on the In Vitro Development of Rat Embryos 109 Experiment 6. Effect of Multiple Exposures to IX Eosin B on the In Vitro Development of Rat Embryos 116 Experiment 7. Effect of Continuous Exposure to IX or 5X Eosin B on the In Vitro Development of Rat Embryos 124 vil Page Experiment 8. Viability of Control or of Rat Embryos Exposed Once to 5X Eosin B and then Transferred to the Left Horn of Naturally Mated Rats 132 Experiment 9. Viability of Control or of Rat Embryos Exposed Once to 5X Eosin B and then Transferred to Both Horns of Naturally Mated Rats 136 Experiment 10. Viability of Control or of Rat Embryos Exposed Once to 5X Eosin B and then Transferred to Opposite Horns of the Same Pseudopregnant Rat 140 Experiment 11. Viability of Control or of Rat Embryos Exposed Once to 5X Eosin B and then Transferred to Both Horns of Pseudopregnant Rats 146 DISCUSSION 152 Effects of Thermal Shock on Embryonic Development and the Staining of Rat Embryos 153 Influence of the Concentration of Eosin B and Stage of Embryonic Development on the Staining Response of Rat Embryos 157 Effects of Single, Multiple, or Continuous Exposure to Eosin B on the In Vitro Development of Rat Embryos 161 Influence of Exposure to Eosin B on the Viability of Rat Embryos Transferred to Naturally Mated or Pseudopregnant Recipient Rats 172 SUMMARY AND CONCLUSIONS 179 REFERENCES 182 ACKNOWLEDGEMENTS 198 viii Page APPENDIX 1 200 Formulation and Storage of Medium for the Culture of Embryos 200 APPENDIX 2 203 Preliminary Trials to Select a Method to Induce Death in Rat Embryos 203 Culture in
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